Spatial pattern of black spot incidence within citrus trees related to disease severity and pathogen dispersal

Guignardia citricarpa, the causal agent of citrus black spot, forms airborne ascospores on decomposing citrus leaves and water-spread conidia on fruits, leaves and twigs. The spatial pattern of diseased fruit in citrus tree canopies was used to assess the importance of ascospores and conidia in citrus black spot epidemics in Sao Paulo State, Brazil. The aggregation of diseased fruit in the citrus tree canopy was quantified by the binomial dispersion index (D) and the binary form of Taylor`s Power Law for 303 trees in six groves. D was significantly greater than 1 in 251 trees. The intercept of the regression line of Taylor`s Power Law was significantly greater than 0 and the slope was not different from 1, implying that diseased fruit was aggregated in the canopy independent of disease incidence. Disease incidence (p) and severity (S) were assessed in 2875 citrus trees. The incidence-severity relationship was described (R-2 = 88.7%) by the model ln(S) = ln(a) + bCLL(p) where CLL = complementary log-log transformation. The high severity at low incidence observed in many cases is also indicative of low distance spread of G. citricarpa spores. For the same level of disease incidence, some trees had most of the diseased fruit with many lesions and high disease severity, whereas other trees had most of the fruit with few lesions and low disease severity. Aggregation of diseased fruit in the trees suggests that splash-dispersed conidia have an important role in increasing the disease in citrus trees in Brazil.

Atmospheric Absorption of Fluoride by Cultivated Species. Leaf Structural Changes and Plant Growth

Fluoride (F) is an air pollutant that causes phytotoxicity. Besides the importance of this, losses of agricultural crops in the vicinity of F polluting industries in Brazil have been recently reported. Injuries caused to plant leaf cell structures by excess F are not well characterized. However, this may contribute to understanding the ways in which plant physiological and biochemical processes are altered. A study evaluated the effects of the atmospheric F on leaf characteristics and growth of young trees of sweet orange and coffee exposed to low (0.04 mol L(-1)) or high (0.16 mol L(-1)) doses of HF nebulized in closed chamber for 28 days plus a control treatment not exposed. Gladiolus and ryegrass were used as bioindicators in the experiment to monitor F exposure levels. Fluoride concentration and dry mass of leaves were evaluated. Leaf anatomy was observed under light and electron microscopy. High F concentrations (similar to 180 mg kg(-1)) were found in leaves of plants exposed at the highest dose of HF. Visual symptoms of F toxicity in leaves of citrus and coffee were observed. Analyses of plant tissue provided evidence that F caused degeneration of cell wall and cytoplasm and disorganization of bundle sheath, which were more evident in Gladiolus and coffee. Minor changes were observed for sweet orange and ryegrass. Increase on individual stomatal area was also marked for the Gladiolus and coffee, and which were characterized by occurrence of opened ostioles. The increased F absorption by leaves and changes at the structural and ultrastructural level of leaf tissues correlated with reduced plant growth.

Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for Paratransgenic Control Strategy of Citrus Variegated Chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.

Genetic Diversity and a PCR-Based Method for Xanthomonas axonopodis Detection in Passion Fruit

Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.

Low glyphosate rates do not affect Citrus limonia (L.) Osbeck seedlings

BACKGROUND: Glyphosate is used to control weeds in citrus orchards, and accidental spraying or wind drift onto the seedlings may cause growth arrest owing to metabolism disturbance. Two experiments were carried out to investigate the effect of non-lethal rates (0, 180, 360 and 720 g Al ha(-1)) of glyphosate on four-month-old `Cravo` lime, Citrus limonia (L.) Osbeck, seedlings. Photosynthesis and the concentrations of shikimic acid, total free amino acids and phenolic acids were evaluated. RESULTS: Only transitory effects were observed in the! contents of shikimate and total free amino acids. No visual effects were observed. CONCLUSION: The present study showed that glyphosate at non-lethal rates, which is very usual when accidental spraying or wind drift occurs in citrus orchard, did not cause severe metabolic damage in `Cravo` lime seedlings. (C) 2009 Society of Chemical Industry

Reclaimed wastewater: Effects on citrus nutrition

The effects of irrigation with reclaimed wastewater (RWW) were compared with well water (WW) on citrus (Citrus paradisi Macfad. X Citrus aurantium L) nutrition. The deviation from the optimum percentage (DOP) index of macro- and micro-nutrients were used to evaluate the nutritional status: optimal (DOP = 0), deficiency (DOP < 0) or excess (DOP > 0). After 11 years of RWW irrigation the influence on nutrient concentration in plants decreased in the order: B > Zn > Mn = Ca > Cu > Mg > P > K. Reclaimed wastewater irritation positively affected citrus nutrition as it rendered the concentration of macronutrients, i.e. P, Ca, and K. closer to their optimum levels (Sigma DOP(macro) = 7). However micro-nutrients tended to be excessive in plants (EDOP(micro) = 753) due to imbalanced supply of these elements in the RWW, particularly, for B and Cu. Citrus groves with long-term RWW irrigation may exercised caution in monitoring concentrations of B and Cu to avoid plant toxicity and soil quality degradation. (C) 2011 Elsevier B.V. All rights reserved.

Sweet Basil (Ocimum basilicum) Extracts Obtained by Supercritical Fluid Extraction (SFE): Global Yields, Chemical Composition, Antioxidant Activity, and Estimation of the Cost of Manufacturing

In this work, supercritical technology was used to obtain extracts from Ocimum basilicum (sweet basil) with CO(2) and the cosolvent H(2)O at 1, 10, and 20% (w/w). The raw material was obtained from hydroponic cultivation. The extract`s global yield isotherms, chemical compositions, antioxidant activity, and cost of manufacturing were determined. The extraction assays were done for pressures of 10 to 30 MPa at 303 to 323 K. The identification of the compounds present in the extracts was made by GC-MS and ESI-MS. The antioxidant activity of extracts was determined using the coupled reaction of beta-carotene and linolenic acid. At 1% of cosolvent, the largest global yield was obtained at 10 MPa and 303 K (2%, dry basis-d.b.); at 10% of cosolvent the largest global yield was obtained at 10 and 15 MPa (11%, d.b.), and at 20% of cosolvent the largest global yield was detected at 30 MPa and 303 K (24%, d.b.). The main components identified in the extracts were eugenol, germacrene-D, epi-alpha-cadinol, malic acid, tartaric acid, ramnose, caffeic acid, quinic acid, kaempferol, caffeoylquinic acid, and kaempferol 3-O-glucoside. Sweet basil extracts exhibited high antioxidant activity compared to beta-carotene. Three types of SFE extracts from sweet basil were produced, for which the estimated cost of manufacturing (class 5 type) varied from US$ 47.96 to US$ 1,049.58 per kilogram of dry extract.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Outcrossing rate in sweet passion fruit based on molecular markers

P>Yellow and sweet passion fruit are insect-pollinated species native to the tropics. Fruits are used commercially for human consumption worldwide. The yellow passion fruit is an outcrossing species with self-incompatible flowers. However, the reproductive system of the sweet passion fruit (Passiflora alata) has not been well elucidated. The objective of this work was to characterize aspects of the mating system in the sweet passion fruit using random amplified polymorphic DNA (RAPD) and microsatellite markers, particularly the rate of outcrossing in P. alata progenies. A multilocus outcrossing rate of t(m) = 0.994 was determined from RAPD and t(m) = 0.940 from microsatellites, supporting P. alata as an outcrossing species. The fixation indices of the maternal generation (F(m)) were -0.200 and 0.071 with RAPD and microsatellite loci, respectively, indicating the absence of inbreeding in the maternal generation. The paternity correlation (r(p)) varied from -0.008 with RAPD markers to 0.208 with microsatellite markers, suggesting a low probability of finding full sibs within the progenies. The results demonstrated that all progenies assessed in this study were derived from outcrossing.

Evaluation of the effects of Candidatus Liberibacter asiaticus on inoculated citrus plants using laser-induced breakdown spectroscopy (LIBS) and chemometrics tools

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents. (C) 2010 Elsevier B.V. All rights reserved.

Venom-Sweet-Venom: N-Linked Glycosylation in Snake Venom Toxins

Protein glycosylation represents one of the most important post-translational events, and is a mean of diversifying a protein without recourse to the genome. The venoms produced by snakes contain an abundance of glycoproteins with N-linked carbohydrates. N-linked glycosylation can ensure the correct folding of important functional domains. Characterization of carbohydrates structures aids in development of human therapeutics by snake venom toxins.

Detection of citrus canker in citrus plants using laser induced fluorescence spectroscopy

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to a large economic loss in citrus production worldwide. In Brazil citrus canker control is done by an official eradication campaign, therefore early detection of such disease is important to prevent greater economic losses. However, detection is difficult and so far it has been done by visual inspection of each tree. Suspicious leaves from citrus plants in the field are sent to the laboratory to confirm the infection by laboratory analysis, which is a time consuming. Our goal was to develop a new optical technique to detect and diagnose citrus canker in citrus plants with a portable field spectrometer unit. In this paper, we review two experiments on laser induced fluorescence spectroscopy (LIF) applied to detect citrus canker. We also present new data to show that the length of time a leaf has been detached is an important variable in our studies. Our results show that LIF has the potential to be applied to citrus plants.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Effects of dietary energy and nitrogen supplements on rumen fermentation and protozoa population in buffalo and zebu cattle

The effects were assessed of two energy sources in concentrate (ground grain corn vs. citrus pulp) and two nitrogen sources (soybean meal vs. urea) on rumen metabolism in four buffaloes and four zebu cattle (Nellore) with rumen cannula and fed in a 4 × 4 Latin square design with feeds containing 60% sugar cane. Energy supplements had no effect on the rumen ammonia concentration in cattle, but ground grain corn promoted higher ammonia level than citrus pulp in buffalo. Urea produced higher ammonia level than soybean meal in both animal species. On average, the buffaloes maintained a lower rumen ammonia concentration (11.7 mg/dL) than the cattle (14.5 mg/dL). Buffaloes had lower production of acetic acid than cattle (58.7 vs. 61.6 mol/100 mol) and higher of propionic acid (27.4 vs. 23.6 mol/100 mol). There was no difference in the butyric acid production between the buffaloes (13.6 mol/100 mol) and cattle (14.8 mol/100 mol) and neither in the total volatile fatty acids concentration (82.5 vs. 83.6 mM, respectively). The energy or nitrogen sources had no effect on rumen protozoa count in either animal species. The zebu cattle had higher rumen protozoa population (8.8 × 10(5)/mL) than the buffaloes (6.1 × 10(5)/mL). The rumen protozoa population differed between the animal species, except for Dasytricha and Charonina. The buffaloes had a lower Entodinium population than the cattle (61.0 vs 84.9%, respectively) and a greater percentage of species belonging to the Diplodiniinae subfamily than the cattle (28.6 vs. 1.4%, respectively). In cattle, ground corn is a better energy source than citrus pulp for use by Entodinium and Diplodiniinae. In the buffaloes, the Entodinium are favored by urea and Diplodiniinae species by soybean meal.

Efeito da associação de sabor e música sobre o estado de ânimo de crianças

Este estudo teve como objetivo avaliar como sabor e música exercem efeito sobre o estado de ânimo de crianças. Participaram 83 crianças de 5 a 10 anos de idade e de ambos os sexos. A tarefa dos participantes consistiu em experimentar o sabor de soluções doce e amarga na ausência de música e na presença de músicas pré-qualificadas como alegres e tristes, e depois, julgar o estado de ânimo decorrente da experimentação. O julgamento do estado de ânimo das crianças se modificou quando o sabor era amargo e as músicas eram alegres, caso em que o estado de ânimo se alterou de triste para alegre; quando o sabor era doce e as músicas tristes, o estado de ânimo passou de alegre para triste. Futuros trabalhos podem observar crianças realizando tarefas que apresentem contextos de alimentos reais associados à estimulação musical.